Electron microscopy and biochemical characterization of a 350-kDa annular hemolymph protein from the keyhole limpet Megathura crenulata.
The isolation and biochemical characterization of an annular non-hemocyanin hemolymph protein from a marine gastropod, the Californian giant keyhole limpet (Megathura crenulata) is presented. By analytical ultracentrifugation, the protein has a sedimentation coefficient of 12S and molecular mass of approximately 350 kDa. The subunit mass, obtained by SDS/PAGE in the presence of -SH reagent and 8 M urea, is approximately 35 kDa, thereby indicating the presence of 10 subunits in the native molecule. By negative staining, the protein is revealed in one predominant image projection as a pentagonal approximately 8 nm ring-like structure with an approximately 2-nm stain-filled centre and, in another image projection, as a dimeric rectangular structure, approximately 8 nm x 14 nm. In deep stain, each of the components of the side-on dimer also shows an indication of stain penetration within a central channel. Unlike the hemolymph protein limulin/C-reactive protein from the horseshoe crab Limulus polyphemus, this Megathura protein does not possess any potential for the aggregation of phosphatidylcholine liposomes although it has an affinity for this phospholipid and causes bilayer destruction. It now appears likely that the previously reported lipid-bilayer ionic-conductance species in keyhole limpet hemolymph, attributed to hemocyanin, could indeed by due to the presence of this annular protein.